Safety Warnings and Precautions For research use only. Not recommended for disease diagnose in humans or animals. Wear gloves when you are handling irritant or harmful reagents. AccuPrep Genomic DNA Extraction Kit Cat. No.: K-3032 Warranty and Liability All BIONEER products are tested under extensive Quality Control procedures. BIONEER guarantees the quality under the warranty period. Any problems should be reported immediately. Liability is conditional upon the customer providing full details of the problem to BIONEER. Once the problem occurs, customer must report to BIONEER within 30 days. Quality Management System ISO 9001 Certified Every aspect of our quality management system from product development, production to quality assurance and supplier qualification meets the world-class standards. QC Testing Each lot of BIONEER s product is carefully tested by the quality control team. Trademarks AccuPrep is a trademark of Bioneer Corporation. Copyright 2004 by Bioneer Corporation. All rights reserved. 1
Contents I. DESCRIPTION... 3 II. KIT COMPONENTS... 4 III. ADDITIONAL REQUIRED MATERIALS... 5 IV. BEFORE YOU BEGIN... 5 V. EXPERIMENTAL PROTOCOL... 6 VI. TROUBLESHOOTING...12 VII. SUPPLEMENTARY MATERIAL...14 AccuPrep Genomic DNA Extraction Kit Technical Manual I. Description AccuPrep Genomic DNA Extraction Kit can rapidly and conveniently extract an average of 6 μg of total DNA from different kinds of sources, such as 200μl of whole blood, 5 x 10 6 leukocytes, mammalian tissues (25~50 mg), or 10 4 ~10 8 cultured cells. AccuPrep Genomic DNA Extraction Kit employs glass fibers, fixed in a column, that specifically binds DNA in the presence of a chaotropic salt. Proteins and other contaminants are eliminated through a series of short wash-and-spin steps. Finally, the genomic DNA is eluted by a low salt solution. This process does not require phenol/chloroform extraction, alcohol precipitation, or other burdensome steps. This kit is suitable to use with whole blood treated with either citrate or EDTA. Advantages: 1. DNA can be extracted promptly and more conveniently. 2. Contaminants, such as proteins and nucleases, which may interfere with PCR reactions, are completely removed, improving the efficiency and reproducibility of PCR. 3. Avoiding precipitation and use of organic solvent, damage to DNA is minimized. 4. The isolated DNA is ready for use in various applications. 3
II. Kit Components This kit is designed to allow 100 preparations and will retain performance for at least one year under standard storage conditions. K-3032 AccuPrep Genomic DNA Extraction Kit Reagents Proteinase K, lyophilized 25 mg X 2 vial One vial with 25 mg of lyophilized proteinase K is included. Dissolve this in 1.25 ml of nuclease-free water. Storage at -20 is recommended to prolong the activity of proteinase K. Please note that repeated freezing and thawing may reduce its activity. Tissue Lysis buffer (TL) 25 ml Mix TL buffer thoroughly by shaking before use. TL buffer is stable for 1 year when stored at room temperature (RT, 15~25 ). Binding buffer (GC) 25 ml Mix GC buffer thoroughly by shaking before use. GC buffer is stable for 1 year when stored at RT. * NOTE: Do not add lyophilized Proteinase K directly to Binding buffer. Washing buffer 1 (W1) 40 ml W1 buffer is supplied in a concentrated form. Before the first use, add 30 ml of absolute ethanol. W1 buffer is stable for 1 year when stored at RT. Washing buffer 2 (W2) 20 ml W2 buffer is supplied in a concentrated form. Before the first use, add 80 ml of absolute ethanol. W2 buffer is stable for 1 year when stored at RT. Elution buffer (EL) 30 ml 10 mm Tris-Cl (ph 8.5). Store at RT. Columns and tubes Binding column tubes 100 ea 2 ml tubes (for filtration) 100 ea 1.5 ml tubes (for elution) 100 ea III. Additional Required Materials 1. Absolute ethanol 2. Absolute isopropanol 3. Table-top microcentrifuge, 10,000 xg (13,000 rpm) 4. Incubator, thermal block, or water bath (shaking) 5. Vortex mixer 6. 1.5 ml tube (for preparation of lysate) 7. Phosphate buffered saline (PBS) IV. Before you begin Before proceeding, please check the following: 1. Did you dissolve proteinase K in 1.25 ml of nuclease-free water? 2. Did you add the correct amount of absolute ethanol to solution W1 and W2? 3. Before starting the extraction process, you must preheat the solution EL to 60. The buffer GC contains irritant chaotropic salt. You should take the appropriate laboratory safety precautions and wear gloves when handling. 4. The g-force can be calculated as follows: rcf = 1.12 x r x (rpm/1,000) 2 Where rcf is the relative centrifugal force (in g), r is the radius of the rotor in centimeters, and rpm is the speed of the centrifuge in revolutions per minute. 4 5
V. Experimental Protocol V-1. Flow chart (for whole blood) V-2. Isolation of DNA from whole blood, buffy coat, and cultured cells. 1. Add 20 μl of Proteinase K (see Before you begin ) to a clean 1.5 ml tube (see Additional required materials ). 2. Apply 200 μl of whole blood, buffy coat or 10 4 ~10 8 cultured cells to the tube containing proteinase K. If the sample volume is less than 200 μl, make the total volume 200 μl by adding PBS. 3. Add 200 μl of Binding buffer (GC) to the sample and mix immediately by vortex mixer. You must completely resuspend the sample to achieve maximum lysis efficiency. 4. Incubate at 60 for 10 min. 5. Add 100 μl of Isopropanol and mix well by pippetting. After this step, briefly spin down to get the drops clinging under the lid. Don t vortex as this might reduce DNA yield. 6. Carefully transfer the lysate into the upper reservoir of the Binding column tube (fit in a 2 ml tube) without wetting the rim. 7. Close the tube and centrifuge at 8,000 rpm for 1 min. You must close each Binding column tube to avoid aerosol formation during centrifugation. If the lysate has not completely passed through the column after centrifugation, centrifuge again at a higher speed (>10,000 rpm) until the binding column tube is empty. 8. Open the tube and transfer the Binding column tube to a new 2 ml tube for filtration (supplied). 6 7
9. Add 500 μl of Washing buffer 1 (W1) without wetting the rim, close the tube, and centrifuge at 8,000 rpm for 1 min. 10. Open the tube and pour the solution from the 2 ml tube into a disposal bottle. 11. Carefully add 500 μl of Washing buffer 2 (W2) without wetting the rim, close the tube, and centrifuge at 8,000 rpm for 1 min. 12. Centrifuge once more at ca. 12,000 rpm for 1 min to completely remove ethanol, and check that there is no droplet clinging to the bottom of Binding column tube. Residual W2 in the Binding column tube may cause problems in later applications. 13. Transfer the Binding column tube to a new 1.5 ml tube for elution (supplied), add 200 μl of Elution buffer (EL, or nuclease-free water) onto Binding column tube, and wait for at least 1 min at RT (15~25 ) until EL is completely absorbed into the glass fiber of Binding column tube. To increase DNA yield, you should wait for 5 min after adding Elution buffer (EL). The volume of EL added can be adjusted from 50 μl to 100 μl. A smaller volume will result in a more concentrated solution, but total yield may be reduced. 14. Centrifuge at 8,000 rpm for 1 min to elute. About 180 μl ~ 200 μl of eluent can be obtained when using 200 μl of Elution buffer (or nuclease-free water). For an improved yield, elute the sample twice and use after concentration process. with Elution buffer (EL) and store at -20, because DNA stored in water is subject to acid hydrolysis. About 6 μg of DNA in 200 μl of eluent (30 ng/μl) with an A 260 /A 280 ratio of 1.6 ~ 1.9 can be typically obtained from 200 μl of whole blood (~ 5 X 10 6 leukocytes/ml). V-3. Isolation of DNA from mammalian tissue. 1. Disrupt (or homogenize) the sample (25~50 mg) with a mortar and pestle, place them in a clean 1.5 ml tube (see Additional required materials ), and add 200 μl of Tissue Lysis buffer (TL). Immediately place the weighted, fresh or frozen tissue in liquid nitrogen and grind to a fine powder with mortar and pestle under liquid nitrogen. Incomplete disruption will lead to significantly reduced yield and can cause clogging of the Binding column tube. The final yield of DNA depends on the amount and the type of tissue used. 2. Add 20 μl of Proteinase K, mix by vortex mixer, and incubate at 60 for 1 hr, or until the tissue is completely lysed. The sample changes in clarity from turbid to clear, indicating that protein digestion has occurred. The time required for lysis will vary depending on the type of tissue used. Lysis will usually take 1~3 hr, and for efficient lysis, a shaking water bath or rocking platform should be used. If these are not available, you should vortex 2~3 times, every 30 min during the incubation. The eluted genomic DNA is stable and can be used directly, or stored at 4 for later analysis. For long-term DNA storage, you should elute 8 9
3. Briefly spin down the tube to remove drops from the inside of the lid and add 200 μl of Binding buffer (GC), and immediately mix by vortex mixer. You must completely resuspend the sample to achieve maximum lysis efficiency. 4. Incubate at 60 for 10 min. 5. Add 100 μl of Isopropanol and mix well by pippetting. After this step, briefly spin down to get the drops clinging under the lid. Don t vortex as this might reduce DNA yield. 6. Carefully transfer the lysate into the upper reservoir of the Binding column tube (fit in a 2 ml tube) without wetting the rim. 7. Close the tube and centrifuge at 8,000 rpm for 1 min. You must close each Binding column tube to avoid aerosol formation during centrifugation. If the lysate has not completely passed through the column after centrifugation, centrifuge again at a higher speed (>10,000 rpm) until the binding column tube is empty. 8. Open the tube and transfer the Binding column tube to a new 2 ml tube for filtration (supplied). 9. Add 500 μl of Washing buffer 1 (W1) without wetting the rim, close the tube, and centrifuge at 8,000 rpm for 1 min. 10. Open the tube and pour the solution from the 2 ml tube into a disposal bottle. 11. Carefully add 500 μl of Washing buffer 2 (W2) without wetting the rim, close the tube, and centrifuge at 8,000 rpm for 1 min. 12. Centrifuge once more at ca. 12,000 rpm for 1 min to completely remove ethanol, and check that there is no droplet clinging to the bottom of Binding column tube. Residual W2 in the Binding column tube may cause problems in later applications. 13. Transfer the Binding column tube to a new 1.5 ml tube for elution (supplied), add 200 μl of Elution buffer (EL, or nuclease-free water) onto Binding column tube, and wait for at least 1 min at RT (15~25 ) until EL is completely absorbed into the glass fiber of Binding column tube. To increase DNA yield, you should wait for 5 min after adding Elution buffer (EL). The volume of EL added can be adjusted from 50 μl to 100 μl. A smaller volume will result in a more concentrated solution, but total yield may be reduced. 14. Centrifuge at 8,000 rpm for 1 min to elute. About 180 μl ~ 200 μl of eluent can be obtained when using 200 μl of Elution buffer (or nuclease-free water). For an improved yield, elute the sample twice and use after concentration process. The eluted genomic DNA is stable and can be used directly, or stored at 4 for later analysis. For long-term DNA storage, you should elute with Elution buffer (EL) and store at -20, because DNA stored in water is subject to acid hydrolysis. About 6 μg of DNA in 200 μl of eluent (30 ng/μl) with an A260/A280 ratio of 1.6 ~ 1.9 can be typically obtained from 200 μl of whole blood (~ 5 X 10 6 leukocytes/ml). 10 11
VI. Troubleshooting 1. There is a low yield or purity of DNA. 1) Buffers or other reagents may have been exposed to conditions that reduce their effectiveness. Ensure that the reagents were stored at room temperature (15~25 C) at all times upon arrival and all reagent bottles were closed tightly after use to preserve ph, stability, and to avoid contamination. After reconstitution of the lyophilized reagents, separate it into aliquots, and store the aliquots at 20. 2) Ethanol may not have been added to the Washing buffer1 and 2. After adding ethanol, mix Washing buffers (W1 and W2) well and always mark the Washing buffer bottles to indicate whether ethanol has been added or not. 3) Reagents and samples may not have been completely mixed. Always mix the sample tube well after adding each reagent. 4) You may not have used the optimal reagent for eluting the DNA. An alkaline ph is required for optimal elution. Use the Elution buffer (EL) included in the kit. 5) The lysis may have been incomplete. Ensure that the sample changes clarity from turbid to clear, indicating that protein digestion has occurred. The time required for lysis will vary depending on the type of tissue used. Lysis will usually take 1~3 hr, and for efficient lysis, a shaking water bath should be used (as directed in the experimental protocol). Mix sample immediately after adding Proteinase K. Always mix the sample thoroughly with isopropanol before adding the lysate onto the Binding column tube. 6) There is a low yield from tissue. Ensure that the tissue was disrupted to small pieces (or a fine powder) before the digestion and lysis steps. There are two following ways to increase the incubation time with Proteinase K: a. Incubate tissue for overnight with Proteinase K. b. Incubate tissue with Proteinase K for 3 ~ 4 hr, then add a fresh aliquot of Proteinase K (30 µl) and incubate for another 1 ~ 2 hr. 7) The absorbance (A 260 ) reading of product is too high. The glass fibers from Binding column tube may have eluted with nucleic acid. These fibers can scatter light, resulting in a higher absorbency reading. In the last stage of elution, too much centrifuge could result in mixing the debris of glass fiber in the binding column tube into the elution. See the below to remove glass fibers 2. There is an incomplete or no restriction enzyme cleavage of isolated DNA. The glass fibers from Binding column tube may have eluted with nucleic acid. These fibers may inhibit enzyme reactions. After the final elution step has been completed, centrifuge at maximum speed for 1 min. Glass fibers may be visible at the bottom of the tube. Transfer the supernatant into a new tube, without disturbing the glass fibers at the bottom of the original tube. 3. DNA from tissue samples is degraded. Tissue should be frozen (-20 C) immediately after harvesting and remain so until the lysis procedure starts. Tissue should be ground to a fine powder with mortar and pestle under liquid nitrogen. There may have been nuclease activity in the unlysed tissue. 12 13
4. The final eluent from blood sample is still slightly colored. The Binding column may have been washed inadequately. Wash the Binding column tube until the flow through is colorless. Repeat the purification protocol by mixing 200 µl of eluent with 200 µl of Binding buffer (GC), then 100 µl of isopropanol. 5. There is a white precipitate in some buffer (TL or GC). A white precipitate may form in Tissue Lysis buffer (TL) or Binding buffer (GC) after prolonged storage at low temperature. Incubating at 60 C should dissolve any precipitate in buffer, TL or GC. The precipitate has no effect on function, and dissolving the precipitate at high temperatures will not improve yield and quality of the purified nucleic acids. VII. Supplementary material 1. Typical Results The yield and purity of genomic DNA varies depending on the sample. The table below shows experimental results. Sample Amount of sample Yield (µg) Whole blood 200 µl 3 ~ 6 Cultured cells 10 6 15 ~ 20 Mammalian tissue 25 ~ 50 mg 10 ~ 15 Yields may vary between different blood donors, because they may have different amounts of leukocytes. 2. Yield of purified DNA 1) You can get about 6 μg of DNA in 200 μl of eluent (30 ng/μl) with an A 260 /A 280 ratio of 1.6 ~ 1.9, from 200 μl of whole blood (~ 5 X 10 6 leukocytes/ml). 2) The yield of DNA depends on both the amount and the type of tissue used. About 25 ~ 50 mg of tissue will yield approximately 15 ~ 20 μg of DNA in 200 μl of eluent (75 ~ 100 ng/μl), with an A 260 /A 280 ratio of 1.6 ~ 1.9. 14 15
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